Bicarbonate Fixation and Malate Compartmentation in Relation to Salt-induced Stoichiometric Synthesis of Organic Acid

The relationship of malate synthesis to K⁺ absorption from solutions of K₂SO₄ and KHCO₃ was compared in nonvacuolate barley (Hordeum vulgare) root tips and whole excised roots. The comparison has permitted separation of the process which evokes organic acid synthesis from that which leads to stoichiometry between net acid equivalents formed and excess K⁺ absorbed from K₂SO₄, on the one hand, and total K⁺ absorbed from KHCO₃, on the other. Both in tips and in roots K⁺ uptake from 20 mN salt solution exceeds malate synthesis in the first hour. In vacuolate roots the expected stoichiometry is achieved with time. When root tips are transferred to dilute CaSO₄, malate is rapidly metabolized, and K⁺ is lost to the solution. By contrast, in excised whole roots the malate level remains unchanged, the salt-induced organic acid presumably being retained in the vacuole. In excised roots malonate leads to a marked drop in malate levels in untreated roots as well as in roots which have experienced salt-induced net malate synthesis. In consequence, it is contended that malonate makes available normally sequestered vacuolar malate.     

An Anomaly in Potassium Accumulation by Barley Roots: II. Effect of Calcium Concentration and Rubidium-86 Labeling

Excised barley roots accumulated 40 to 50% more K⁺ from 0.04 mm than from 0.06 mm KCl when incubated for 24 hours in KCl solutions containing 0.2 mm CaSO₄. This phenomenon was not markedly influenced by the rate of absorption of the counteranion. The presence of Na⁺ in the treatment solutions decreased total K accumulation but did not alter the K⁺ concentration at which the accumulation peak occurred. Short interval studies indicated that this phenomenon is easily observable after 4 hours and begins to become apparent within 2 hours. In comparison with barley, accumulation of K⁺ by excised wheat roots decreased as KCl concentration was increased from 0.02 to 0.06 mm; but K⁺ accumulation curve for corn roots showed no peaks or depressions in the concentration range of 0.01 to 0.1 mm. A normal hyperbolic curve was noted for the accumulation of Na⁺ from 0.01 to 1 mm NaCl by barley roots.     

Short-term experiments on ion transport by seedlings and excised roots : technique and validity

The absorption of K⁺ by excised roots of barley (Hordeum vulgare L. cv California Mariout) has been systematically compared with that of entire, undisturbed seedlings. Some experiments have also been done with wheat (Triticum aestivum L.) and an amphiploid obtained from a cross between it and salt-tolerant tall wheatgrass (Lophopyrum elongatum Host Löve [syn. Agropyron elongatum Host]). For all three genotypes, the rate of K⁺ absorption measured in a 20-min period was identical for entire 8-d-old seedlings and their excised roots within the experimental error. Manipulation gentler than root excision, viz. careful transfer of seedlings from one experimental solution to another, was also without effect on the rate of K⁺ absorption. Absorption of K⁺ measured by assay of its ⁸⁶Rb label in the tissue was identical with that measured by K⁺ depletion of the experimental solutions assayed chemically. For the plant materials and conditions of these experiments, the excised root technique for studying ion transport into roots is validated. The advantages of the technique, and findings differing from the present ones, are discussed.     

Calcium inhibition of potassium absorption in corn roots

Calcium (or magnesium) sulfate or chloride was found to inhibit energy dependent potassium transport in excised corn roots. This Ca²⁺ inhibition of K⁺ transport was most pronounced during the initial phases of transport. As the absorption periods were lengthened the effect of Ca²⁺ gradually changed from an inhibition to a typical promotion (after about 30-45 mins) of K⁺ transport. Kinetic analysis indicated the inhibition to be of a non-competitive nature.

Identical experiments with excised barley roots showed that CaSO₄ had no effect on K⁺ absorption whereas CaCl₂ had a typical stimulatory effect on K⁺ absorption. Kinetic analysis indicated that both corn and barley have efficient K⁺ transporting systems but barley roots are approximately 5 times more active (on a fr wt basis) than corn roots.

These results illustrate the hazards involved in applying results obtained with 1 (or even several) plant species to all species.

     

The influence of potassium content on the kinetics of potassium influx into excised ryegrass and barley roots

The influx of K⁺ into excised roots of barley (Hordeum vulgare L.) and ryegrass (Lolium multiflorum L.) previously grown with or without K⁺ was measured in K⁺ solutions ranging in concentration from 0.01 to 50 mM. In both species the K⁺ influx was lower in the roots with high K⁺ content. The extent of reduction by high internal [K⁺] decreased with external concentration above 1 mM. These results support the contention that at high external concentrations passive diffusion makes significant contributions to observed fluxes.     

Effect of calcium on the absorption and translocation of heavy metals in excised barley roots: Multi-compartment transport box experiment

Summary The effect of Ca on the absorption and translocation of Mn, Zn and Cd in excised barley roots was studied using a multi-compartment transport box technique. A radioisotope (⁵⁴Mn,⁶⁵Zn or^115mCd)-labelled test solution was supplied to the apexes of excised roots and the distribution pattern in the roots was examined in the absence or presence of Ca. Results obtained were as follows. Addition of Ca to the test solution reduced the absorption of Mn and inhibited drastically its translocation in excised roots. With increasing concentrations of Ca in test solutions, its inhibitory effects on the absorption and translocation of Mn became severe. Similar results were observed for the absorption and translocation of Zn. Ca in the test solution decreased the absorption and inhibited drastically the translocation of Zn; as in the case of Mn, higher concentrations of Ca had severe effects on these functions. It was also evident that the addition of Ca to the test solution reduced the absorption of Cd at all levels of Cd concentration (1, 10, and 100 μM). Cd absorption decreased with increasing concentrations of Ca in the test solution. However, Ca accelerated the translocation of Cd in excised roots supplied with test solutions containing up to 10μM Cd. At 100μM Cd, addition of Ca caused a negligibly small acceleration of Cd translocation. The accelerating effect of Ca on Cd translocation, especially “xylem exudation”, decreased markedly with the addition of 2,4-dinitrophenol, but not with the addition of chloramphenicol or p-chloromercuribenzene sulphonic acid. When barley plants were supplied with only CaSO₄ during the entire growing period, that is, plants were not supplied with nutrient solution on the last day of this period, Ca had no accelerating effect on Cd translocation in excised roots.     

Cation Effects on Chloride Fluxes and Accumulation Levels in Barley Roots

Accumulation of Cl^- by excised barley roots, as of K⁺, approaches a maximum level at which the ion influx and efflux rates become equal. The rate of Cl^- influx at this equilibrium is close to the initial rate while the efflux rate increases with time from zero to equality with influx. The Cl^- fluxes are independent of simultaneous exchange flux of the cations, but depend on the nature and concentration of the salt solutions from which they originate. The Cl^- content at equilibrium, however, is largely independent of the external concentrations. The approach to equilibrium reflects the presence of the cation. Cl^- flux equilibrium is attained more rapidly in KCl than in CsCl or CaCl₂. This is presumably an effect of much slower distribution of Cs⁺ and Ca⁺⁺ than of K⁺ within the roots. Accumulated Cs⁺ appears to form a barrier to ion movement primarily within the outermost cells, thereby reducing influx and ultimately efflux rates of both Cl^- and cations. Slow internal mixing and considerable self-exchange of the incoming ions suggest internal transport over a series of steps which can become rate-limiting to the accumulation of ions in roots.     

Influence of Excision and Aging upon K+ Influx into Barley Roots: Recovery or Enhancement? 1

The influx of K⁺ from ⁸⁶Rb-labeled solutions in the concentration range 0.008 to 0.2 mm into roots of intact plants and excised roots of barley plants (Hordeum vulgare [L.]) previously grown in 5 mm CaSO₄ (low K⁺ roots) or 0.5 mm CaSO₄ plus 5 mm KCl (high K⁺ roots) was measured. A consistent observation of these experiments was a substantial reduction of influx (usually by about 50%) following excision. The possible leakage of K⁺ into the medium and subsequent dilution of specific activity of labeled solutions was eliminated as an explanation for influx reduction in excised low K⁺ roots. Reduction of transpirational rates was also without effect upon influx into low K⁺ roots. Excision followed by 2 hours aging in 0.5 mm CaSO₄ solution revealed that influx values recovered within the 2 hours to the values obtained in intact roots. It is concluded that much of the literature which describes the enhancement of ion uptake following excision actually describes excision damage followed by recovery.     

Cation-Anion Balance during Potassium and Sodium Absorption by Barley Roots

Steady-state rates of potassium ion and sodium ion absorption by excised barley roots accompanied by various anions were compared with the rates of anion absorption and the concomitant H⁺ and base release by the roots. The cation absorption rates were found to be independent of the identities, concentrations, and rates of absorption of the anions of the external solution, including bicarbonate. Absorption of the anion of the salt plus bicarbonate could not account for the cation absorption. H⁺ is released during cation absorption and base during anion absorption. The magnitude by which one or the other predominates depends on the relative rates of anion and cation absorption under various conditions of pH, cation and anion concentration, and inhibitor concentrations. The conclusion is that potassium and sodium ions are absorbed independently of the anions of the absorption solution in exchange for H⁺, while anions are exchanged for a base. The H⁺ release reflects a specificity between K⁺ and Na⁺ absorption such that it appears to be H⁺ exchanged in the specific rate-limiting reactions of the cation absorption.     

Potassium transport in salt-stressed barley roots

Salinization of the medium inhibits both K⁺ uptake by excised barley (Hordeum vulgare L.) roots and K⁺ release from their stele, as measured by short-term ⁸⁶Rb uptake and xylem exudation, respectively. Although inhibition was not specific to chloride, mannitol caused a different response from that of inorganic sodium salts, indicating that inhibition was at least partly the result of an ion effect. In roots previously exposed to low levels of NaCl, NaCl stress directly affected stelar K⁺ release, whereas in low-sodium roots stelar K⁺ release was much less salt-sensitive than K⁺ uptake.Abbreviation chCl choline chloride     

K+-dependent net Na+ efflux in roots of barley plants

Summary The influence of K⁺ ions on the net Na⁺ fluxes in cells of excised barley roots (Hordeum distichon L.) and roots of whole barley plants was investigated. The fluxes were determined by flame photometry in the external solution. In both cases a transient net Na⁺ efflux against the external Na⁺ concentration was observed upon addition of K⁺. The results stress the effectiveness of the K⁺-dependent Na⁺ efflux mechanism residing at the plasmalemma, and its involvement in K–Na-selectivity in whole barley plants.     

Equilibrium and Ion Exchange Characteristics of Potassium and Sodium Accumulation by Barley Roots

Potassium ion and Na⁺ influx and efflux rates into and from excised barley roots are compared with the maximum capacity of accumulation. Potassium ion and Na⁺ influx and efflux involve a cation exchange that is independent of simultaneous exchange of the accompanying anion. These exchange fluxes depend on the concentration and cation composition of the solutions from which they originate. Selective differences between K⁺ and Na⁺ fluxes are sufficient to account for a cationic distribution within the roots that differs markedly from that of the external solution and that persists for extended time periods. The accumulation maximum is a cation exchange equilibrium with the cation influx and efflux rates approaching equality. The equilibrium level is independent of the individual cation fluxes and the external solution concentration. It is a finite quantity which appears to be determined by the internal anion concentration including accumulated as well as endogenous anions.     

Short Report : Effect of amines and guanidines on Rb+ absorption by excised corn roots

The effect of alkyl-amines and -guanidines on the absorption of rubidium by the excised roots of the corn plant was tested. Inhibition of Rb⁺ absorption was observed with both amines and guanidines, where guanidines were more effective. The effect of alkylamines on Rb⁺ transport depends on their molecular structure.     

Boron uptake by excised barley roots: I. Uptake into the free space

At 2 C, all boron accumulated by excised barley roots (Hordeum vulgare L. cv. Herta) remains in the free space; i.e. active uptake is nil at this temperature. Three component fractions of free space B were apparent: (a) a surface contaminant film of B on blotted roots, (b) water free space B, and (c) B reversibly bound in the cell walls. A stoichiometric release of H⁺ from the roots in the presence of B indicated that B was bound by borate complexes with polysaccharides in the cell walls. Polysaccharide-borate complexes are much less stable than those of monosaccharides, and the bound B fraction could be readily removed by rinsing the roots in the presence of a monomeric polyol possessing the necessary cis-diol configuration. Cell wall material separated from excised barley roots had a B binding capacity 66% greater than that of intact roots.     

Mesurement of longitudinal ion profiles in single roots of Hordeum and Atriplex by use of flameless atomic absorption spectroscopy

Summary A method is described by which the Na⁺ and K⁺ content in 0.5 mm sections of single roots of Hordeum distichon L. and Atriplex hortensis L. can be determined by use of flameless atomic absorption spectroscopy. By this method the longitudinal profiles of K⁺ and Na⁺ along low salt roots and roots which had been equilibrated with or grown in K⁺-free 1 mM Na⁺-solution were determined. The profiles reveal that high K⁺/Na⁺ ratios in the cytoplasm are maintained also in K⁺-free solutions. In solutions containing 1 mM Na⁺ a high K⁺/Na⁺ selectivity was found to be dependent on sufficient aeration. From the ion profiles the cytoplasmic (110 mM) and vacuolar (20 mM) K⁺ concentration in low salt barley roots—values which are unobtainable by compartmental analysis—could be estimated.     

Potassium and sodium absorption kinetics in roots of two tomato species : lycopersicon esculentum and lycopersicon cheesmanii

Excised roots of the tomato species, Lycopersicon esculentum Mill. cv Walter (the commercial species) and of Lycopersicon cheesmanii ssp. minor (Hook.) C.H. Mull. (a wild species from the Galapagos Islands), were used in comparative studies of their absorption of K⁺ and Na⁺. Uptake of ⁸⁶Rb-labeled K⁺ and ²²Na-labeled Na⁺ by excised roots of `Walter' and L. cheesmanii varied as a function of genotype and tissue pretreatment with or without K<sup>+</sup>. Excised roots of `Walter' consistently absorbed more ⁸⁶Rb-labeled K⁺ than those of L. cheesmanii. Absorption of K⁺ from solutions ranging from 0.01 to 0.2 millimolar KCl showed saturation kinetics in both K⁺-pretreated and K⁺-depleted roots of `Walter,' and for K<sup>+</sup>-depleted roots of L. cheesmanii. K<sup>+</sup>-pretreated roots of L. cheesmanii had exceedingly low rates of K<sup>+</sup> uptake with strikingly different, linear kinetics. Pretreatment with K<sup>+</sup> caused a decrease in rates of K<sup>+</sup> uptake in both genotypes. Potassium depleted roots of L. cheesmanii absorbed Na<sup>+</sup> at a greater rate than those of `Walter,' whereas K⁺-pretreated roots of `Walter' absorbed Na⁺ at a greater rate than those of L. cheesmanii. The results confirm and extend previous conclusions to the effect that closely related genotypes may exhibit widely different responses to the two alkali cations, K⁺ and Na⁺.     

Regulation of Plasma Membrane H+-Pump Activity by 14-3-3 Proteins in Barley (Hordeum disticum) Roots under Salt Stress

Regulatory changes in the activity of the plasma membrane H⁺-ATPase in salt-stressed roots were investigated using seven-day-old seedlings of two cultivars of barley (Hordeum disticum L.) with different salt tolerances: Moskovskii-121 (salt-tolerant) and Elf (salt-sensitive). During the first hour of salt stress, the rate of proton extrusion from the excised roots increased in parallel with the ATP hydrolase activity and the amount of 14-3-3 proteins bound to H⁺-ATPase in isolated plasma membranes. Subsequently, all these parameters decreased and dropped after 3–6 h below the initial levels. The initial stimulation of proton extrusion from the detached barley roots was caused by osmotic stress, whereas the subsequent retardation of proton extrusion was probably caused by a toxic effect of excessive Na⁺ content in the cytoplasm. The salt-stress responses showed similar trends in both cultivars, with the exception that Moskovskii-121 responded faster than cv. Elf. The results indicate that 14-3-3 proteins regulate the H⁺-ATPase activity in the plasma membranes of barley root cells during salt stress; furthermore, the response time might be a useful indicator to discriminate cultivars with different salt tolerances.     

Temperature and pH interaction in NO3 uptake

Summary The interaction effects of temperature, pH and the presence of the poly-valent cation aluminium on nitrate uptake by excised barley roots have been studied. Results indicate that nitrate uptake increased with temperature at the two pH values studied. NO₃ ⁻ uptake was higher from pH6 than from pH4 at all temperatures in the absence of Al⁺³. The addition of Al⁺³ to the external solution modifies this effect. Different theories are given to explain these interactions.     

Adaptation of barley roots to low oxygen supply and its relation to potassium and sodium uptake

The uptake of Na⁺ and K⁺ by barley seedlings grown on aerated or non-aerated solutions was studied. Plants growing in culture solution took up K⁺ with high selectivity whether the solution was aerated or not. Roots of plants grown on aerated CaSO₄ and transferred to a solution of KCl and NaCl had a lower preference for K⁺ than roots of plants grown on non-aerated CaSO₄. Both kinds of low-salt roots were much less able to discriminate between K⁺ and Na⁺ than high-salt roots grown on a culture solution. The different levels of K⁺ selectivity are suggested to be related to H⁺ release from the tissue.     

Vanadium Uptake by Plants: Absorption Kinetics and the Effects of pH, Metabolic Inhibitors, and Other Anions and Cations

The kinetics of vanadium absorption by excised barley (Hordeum vulgare L., cv. Eire) roots were investigated with respect to ionic species of V in solution, time and concentration dependence, Ca sensitivity, and interaction with various anions, cations, and pH levels. The role of metabolism in V absorption was also studied using anaerobic treatment (N₂ gas) and chemical inhibitors (NaN₃, KCN, or 2,4-dinitrophenol). Approximately one-third of the labeled V initially taken up by excised roots was desorbed to a constant level after 45 min in unlabeled V solutions. The rate of absorption of labeled V from 5 μm NH₄VO₃ solutions containing 0.5 mm CaSO₄ was constant for at least 3 hours. Omission of Ca resulted in a 72% reduction in V uptake when compared to controls with 0.5 mm CaSO₄. The rate of uptake of V was highest at pH 4 but dropped to a very low level at pH 10. It was relatively constant between the pH levels of 5 and 8 at which the VO₃⁻ ion is the predominant ionic species in solution. The rate of absorption of V was followed as a function of concentrations from 0.5 to 100 μm NH₄VO₃. It was found to be a linear function of concentration and did not follow saturation kinetics. Absorption experiments carried out with labeled V from either N_aVO₃ or NH₄VO₃ sources gave similar results. No anion studied (i.e. HPO₄²⁻, HAsO₄²⁻, MoO₄²⁻, SeO₄²⁻, SeO₃²⁻, CrO₄²⁻, BO₃³⁻, No₃⁻, and Cl⁻) interfered appreciably (i.e. less than 30% inhibition) with the absorption of labeled V. Anaerobic treatment of absorption solution with N₂ gas did not inhibit V absorption by excised roots. The results obtained using chemical inhibitors were not consistent. It was concluded that V is not actively absorbed by excised barley roots.